Most semisynthetic .beta.-lactam antibiotics are prepared by reaction of 6-aminopenicillanic acid (6-APA) or 7-aminodesacetoxycephalosporanic acid (7-ADCA) with the required side-chain precursor. 6-APA is typically produced by enzyme based hydrolysis of either benzyl penicillin (PenG) or phenoxymethyl penicillin (PenV). 7-ADCA is prepared by enzymatic hydrolysis of cephalosporin G, which is available by ring expansion of PenG. ##STR1##
Enzyme processes used for the manufacture of 6-APA or 7-ADCA rely on the use of enzyme which has been immobilized on a suitable support. This makes separation of the reaction products from the biocatalyst a simple procedure. The reaction is carried out in aqueous media at elevated temperatures (e.g. 28.degree.-37.degree. C.). Several different reactor configurations have been described in the literature, with stirred tank reactors and packed bed reactors being the most popular. In almost all cases, the enzyme is immobilized on an inert support of some type so that it can be used for multiple batches. In the case of the stirred tank batch reactor, substrate penicillin (in the form of its potassium salt) is charged to a large vessel containing the immobilized enzyme. The reaction pH is maintained in the desired range through the controlled addition of a weak base such as dilute ammonium hydroxide or caustic. The reaction is allowed to proceed to 95-99% conversion, at which point the immobilized enzyme is filtered off and the 6-APA/acid/water filtrate discharged to a downstream recovery operation. The crystalline 6-APA product is precipitated by the addition of inorganic acid and recovered by simple filtration and drying. The phenylacetic acid (in the PenG case) or the phenoxyacetic acid (in the case of PenV) is extracted into an organic solvent and recycled back upstream in the process to the penicillin fermentation step. These immobilized enzyme processes have been in use for over 20 years.
In the early development of commercial 6-APA processes, the need to immobilize the enzyme was necessitated in part by the cost of the enzyme which had to be reused many times in order to make its cost contribution to the final product as low as possible. By immobilizing the enzyme on a solid support, the separation of the aqueous product solution from the catalyst became simply a matter of passing the reactor contents through filters capable of retaining the enzyme support. The size difference between the products and the catalyst is in the order of 10.sup.5 when the catalyst is immobilized, so that crude filters would suffice.
More recently it has been recommended that enzymes be immobilized on the surface of an ultrafiltration membrane. See, for example, European patent 69869, U.S. Pat. No. 4,800,162 and Bryjak and Noworyta [Biochem. Eng.--Stuttgart, Proc. Int. Symp. 2nd 1990, 122-125 ed. Reuss; Fischer Stuttgart (1991)]. The process is operated such that product ultrafiltration is done on a continuous basis. The major flaw in this configuration is that the rate of continuous addition of substrate must be relatively small to ensure that high conversions are obtained. This type of configuration is called a Continuous Stirred Tank Reactor (CSTR). To overcome the limitations of the CSTR configuration, a number of tanks are connected in series, which requires more tanks, membrane area, pumps, and pH control equipment.
While these approaches offer certain advantages, drawbacks remain: (1) there is always some, and often much, inactivation of the enzyme as a result of the immobilization chemistry; (2) pH control and diffusion resistance become significant concerns because of restricted access to the enzyme; (3) a considerable volume of the reactor is consumed by the inert carrier; and (4) immobilization often requires expensive chemistry, more time and more sophisticated equipment. There is thus a need for a process that minimizes or eliminates the foregoing drawbacks.